Studies on the structure and function of two enzymes involed in the metabolism of Vitamin B6 in brain will continue with emphasis on 1) the mechanism of the reaction catalyzed by pyridoxal kinase. The binding of the substrates, pyridoxal, pyridoxine and ATP to the catalytic site will be investigated by absorption and fluorescence methods. Identification of the amino acid residues responsible for binding the substrates and the amino acid residues which participate in the catalytic steps. Investigation of the reactivity of the enzymes from liver and brain with antibodies by using radioimmunoassay techniques. 2) Characterization of P-pyridoxine oxidase from brain; mechanisms of binding of the cofactor FMN to the apoprotein and interaction of the cofactor with the protein subunits. Kinetic methods as well as other spectroscopic methods, i.e., resonance energy transfer, and emission anisotropy, will be used to detect interactions between pyridoxal kinase and P-pyridoxine oxidase. With these approaches we should attempt to answer a fundamental question: "What are the mechanisms regulating the concentration of P-pyridoxal in brain?" 3) Structure and function of 4-aminobutyrate aminotransferase: the rate of the reaction between the substrates, 4-aminobutyrate, beta-analine, sigma-aminovaleric and the P-pyridoxal form of the enzyme will be studied by rapid mixing techniques in an effort to gain more information about the transamination reaction. P3' nuclear magnetic resonance spectroscopy will be used to gain information about the environment of the essential phosphate group of the cofactor. This technique, together with absorption and emission spectroscopy, will provide information about the microenvironment of the active site.